1. Field of the Invention
This invention is in the field of biology and more particularly in the field of cell biology.
2. Description of the Prior Art
The ability to grow mammalian cells is important at both the laboratory and industrial levels. At the laboratory level, the limiting factor for cellular or viral research at the sub-cellular level is often the amount of raw material available to be studied. At the industrial level, there is much effort being devoted to the development of pharmaceuticals based on mammalian cell products. These are primarily vaccines for human or animal viruses, but also include human growth hormone and other body hormones and biochemicals for medical applications.
Some mammalian cell types have been adapted for growth in suspension cultures. Examples of such cell types include HeLa (human), BHK (baby hamster kidney) and L cells (mouse). Such cells, in general, have non-normal genetic complements, i.e., too many or too few chromosomes or abnormal chromosomes. Often, these cells will produce a tumor upon injection into an animal of the appropriate species.
Other mammalian cell types have not been adapted for growth in suspension culture to date, and will grow only if they can become attached to an appropriate surface. Such cell types are generally termed "anchorage-dependent" and include 3T3 mouse fibroblasts, mouse bone marrow epithelial cells; Murine leukemia virus-producing strains of mouse fibroblasts, primary and secondary chick fibroblasts; WI-38 human fibroblast cells; and, normal human embryo lung fibroblast cells (HEL299, ATCC #CCL137). Some anchorage-dependent cells have been grown which are tumor causing but others were grown and found to be non-tumor causing. Also, some anchorage-dependent cells, such as WI-38 and HEL299, can be grown which are genetically normal.
Whereas considerable progress has been made in large scale mammalian cell propagation using cell lines capable of growth in suspension culture, progress has been very limited for large scale propagation of anchorage-dependent mammalian cells. Previous operational techniques employed for large scale propagation of anchorage-dependent cells were based on linear expansion of small scale processes. Cell culture plants utilized a large number of low yield batch reactors, in the forms of dishes, prescription bottles, roller tubes and roller bottles. Each of these was a discrete unit or isolated batch reactor requiring individual environmental controls. These controls, however, were of the most primitive type due to economic considerations. Variation in nutrients was corrected by a medium change, an operation requiring two steps, i.e., medium removal and medium addition. Since it was not uncommon for a moderately sized facility to operate hundreds of these batch reactors at a time, even a single change of medium required hundreds of operations, all of which had to be performed accurately, and under exacting sterile conditions. Any multiple step operation, such as cell transfer or harvest, compounded the problem accordingly. Thus, costs of equipment, space and manpower were great for this type of facility.
There are alternative methods to linear scale-up from small batch cultures which have been proposed. Among such alternatives which have been reported in the literature are plastic bags, stacked plates, spiral films, glass bead propagators, artificial capillaries, and microcarriers. Among these, microcarrier systems offer certain outstanding and unique advantages. For example, great increases in the attainable ratio of growth surface to vessel volume (S/V) can be obtained using microcarriers over both traditional and newly developed alternative techniques. The increase in S/V attainable allows the construction of a single-unit homogeneous or quasi-homogeneous batch or semi-batch propagator for high volumetric productivity. Thus, a single stirred tank vessel with simple feedback control for pH and pO.sub.2 presents a homogeneous environment for a large number of cells thereby eliminating the necessity for expensive and space consuming, controlled environment incubators. Also, the total number of operations required per unit of cells produced is drastically reduced. In summary, microcarriers seem to offer economies of capital, space and manpower in the production of anchorage-dependent cells, relative to current production methods.
Microcarriers also offer the advantage of environmental continuity since the cells are grown in one controlled environment. Thus, microcarriers provide the potential for growing anchorage-dependent mammalian cells under one set of environmental conditions which can be regulated to provide constant, optimal cell growth.
One of the more promising microcarrier systems to date has been reported by van Wezel and involves the use of diethylaminoethyl (DEAE)-substituted dextran beads in a stirred tank. A. L. van Wezel, "Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture", Nature 216:64 (1967); D. van Hemert, D. G. Kilburn and A. L. van Wezel, "Homogeneous Cultivation of Animal Cells for the Production of Virus and Virus Products", Biotechnol. Bioeng. 11:875 (1969); and A. L. van Wezel, "Microcarrier Cultures of Animal Cells", Tissue Culture, Methods and Applications, P. F. Kruse and M. K. Patterson, eds., Academic Press, New York, p. 372 (1973). These beads are commercially produced by Pharmacia Fine Chemicals, Inc., Piscataway, N.J., under the tradename DEAE-Sephadex A50, an ion exchange system. Chemically, these beads are formed from a crosslinked dextran matrix having diethylaminoethyl groups covalently bound to the dextran chains. As commercially available, DEAE-Sephadex A50 beads are believed to have a particle size of 40-120.mu.m and a positive charge capacity of about 5.4 meq per gram of dry, crosslinked dextran (ignores weight of attached DEAE moieties). Other anion exchange resins, such as DEAE-Sephadex A25, QAE-Sephadex A50 and QAE-Sephadex A25 were also stated by van Wezel to support cell growth.
The system proposed by van Wezel combines multiple surfaces with movable surfaces and has the potential for innovative cellular manipulations and offers advantages in scale-up and environmental controls. Despite this potential, these suggested techniques have not been significantly exploited because researchers have encountered difficulties in cell production due to certain deleterious effects caused by the beads. Among these are initial cell death among a high percentage of the cell inoculum and inadequate cell growth even for those cells which attach. The reasons for these deleterious effects are not thoroughly understood, although it has been proposed that they may be due to bead toxicity or nutrient adsorption. See van Wezel, A. L. (1967), Nature 216: 64-65; van Wezel, A. L. (1973), Tissue Culture, Methods and Applications. Kruse, P. R. and Patterson, M. R. (eds.), pp. 372-377, Academic Press, New York; van Hemert, P., Kilburn, D. G., and van Wezel, A. L. (1969), Biotechnol. Bioeng. 11: 875-885; Horng, C. and McLimans, W. (1975), Biotechnol. Bioeng. 17: 713-732.
It could be that the deleterious effects of these commercially available ion exchange resins are due to their method of manufacture. Certain of these production methods are described for polyhydroxy materials in patents such as: U.S. Pat. Nos. 3,277,025; 3,275,576; 3,042,667 and 3,208,994 all to Flodin et al. Whatever the reason, however, the presently commercially available materials are simply not sufficient for good cell growth of a wide variety of cell types.
One solution to overcoming some of the deleterious effects encountered in attempts to use such commercially available microcarriers for cell growth is described in U.S. Pat. No. 4,036,693, issued on July 19, 1977 to Levine et al. Therein, a method for treating these commercially available ion exchange resins with macromolecular polyanions, such as carboxymethylcellulose, is proposed. While this method has proven successful, it would clearly be more advantageous if the beads could be manufactured initially to have properties designed for outstanding growth of anchorage-dependent cells.